Human skin is a unique tissue and the human body's largest organ. The skin is composed of two layers; the epidermis is the outermost relatively thin layer; and the dermis is the inner relatively thick layer. The two layers are bound together by the basal lamina. The epidermis forms a barrier whose primary function is protective and it is also essential to life. It is composed of four general sub-layers; the stratum germinativum or basal layer (the most inner layer and whose cells abut the basallamina; the stratum spinosum, the stratum granulosum and the stratum corneum, the outermost layer). Each of these layers is derived from cells of the preceding layer, with new cells generated from the basal layer, which contains mitotically active keratinocytes and stem cells. The first three layers all serve to create the stratum corneum, which is the functional barrier for water retention, pathogen exclusion and protection from chemical and or physical elements in the environment. A normal healthy epidermis renews itself on average every 30 days.
The outer and essential stratum corneum is produced as the end product of an orchestrated suite of keratinocyte differentiation and differential gene expression, leading to terminal differentiation and cell death. The stratum corneum is therefore composed of enucleate, “dead” cells. If the process of differentiation of cells to produce the stratum corneum is perturbed, the consequences can be dramatic, resulting in any of a variety of diseases, many of which are fatal.
Key to the biology of the epidermis and the production of a healthy stratum corneum is differential gene expression, which precedes and directs keratinocyte differentiation. Because changes in gene expression precede or accompany changes in skin physiology, monitoring gene expression in the skin in health and disease is of particular medical interest. Changes in gene expression are conveniently measured as changes in messenger RNA (mRNA) levels or specific proteins within the cell.
Traditional methods for the recovery of nucleic acids (RNA and DNA) and proteins from the skin are invasive, i.e. the punch or shave biopsy. These sampling methods require the use of a scalpel or other sharp instrument (e.g. dermatome, curette) to recover a tissue specimen. These sampling methods invariably isolate epidermis as well as dermis, require local anesthesia, create bleeding wounds, produce scarring and in the case of biopsies greater than 3 mm, generally require sutures to close the skin. Biopsy sites represent direct access for pathogens to the body and thus are sites of potential internal and superficial (skin) infection to healthy individuals. People who are immunocompromised or diabetic and have impaired wound healing may be at higher risk for infections from biopsy procedures. However, the information gained from biopsy (e.g. diagnosis of disease, physiological data) typically outweighs the slight risk that biopsy presents. Nonetheless, any method that can produce similar information but avoids wounding and the risk of infection would be preferable to biopsy.
Before 1999 there were few alternatives to skin biopsy if the goal was to isolate a skin sample for molecular analysis, such as DNA or RNA analysis. Use of the polymerase chain reaction in quantification of interleukin 8 mRNA in minute epidermal samples utilized curettage (scraping) to isolate superficial skin samples for subsequent analysis by reverse transcription PCR analysis (RT-PCR) of IL-8 mRNA expression. However, scraping is highly dependant on the skill of the operator if bleeding is to be avoided and the method has never been used as a routine technique for skin sampling. Thus until 1999 there was no simple and non-invasive method of procuring a sample for the analysis of RNA expression in the skin.
In 1999 it was demonstrated that a well known method of superficial skin removal, tape stripping, could be used to recover RNA from the skin. It was shown that tape strip recovered RNA could be used to differentiate irritant from allergic contact dermatitis. Unfortunately, this method was not practical because it usually took more than 20 applications of fresh tape to recover a sample.
In 2004 it was demonstrated that the first four applications of tape were sufficient in most cases to obtain an RNA sample. The recovered RNA was capable of analysis by RT-PCR and DNA microarray technologies. The method has also been utilized to collect RNA for RT-PCR analysis of RNA from psoriatic lesions. Further work by researchers in this field demonstrated that recovery of RNA from the skin was (i) reproducibly variable; (ii) that recovery varied significantly at different anatomical locations on the same individual; and (iii) that recovery varied between individuals at similar anatomical locations.
The variability of RNA recovery by tape stripping resembles the variability in yield of stratum corneum by tape stripping and the variation in recovery of topically applied drugs. The source of this variation is believed to lie in the nature of the skin surface as skin is irregular, with natural crevasses, contours, and wrinkles all conspiring to create a rough and variable surface. Features such as hair follicles, sweat and sebaceous ducts also create “holes” in the stratum corneum that contribute to additional surface irregularity. It has been demonstrated that adhesive tape, with its limited ability to conform to microscopic structures cannot mold itself to surface irregularities and thus cannot remove a uniform layer of skin. Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin. Repeated tape stripping does not immediately solve this problem.
An additional deficiency to the use of adhesive tape to recover skin samples (hence RNA) is the variability in adhesion to the skin encountered with different operators. It has been reported that differing applied pressures and methods of applying pressure result in different degrees of adhesion to the skin and consequentially differential removal of a sample. While some groups have mitigated this deficiency by applying a measured amount of force when applying tape in experimental situations, this added step would be an encumbrance in a clinical situation. These encumbrances are clearly revealed by the preponderance of published clinical trials employing tape stripping where method and applied pressure are uncontrolled variables.
The use of adhesive tape is further made inconvenient in a clinical and commercial setting by restrictions imposed by the physical dimensions of the tape itself. By necessity tape comes in a particular size; however, skin lesions come in a range of sizes and shapes. When faced with a tape of particular dimensions an operator must pay particular attention to applying the tape to the lesion alone and avoiding the surrounding non-lesional skin. This lesion-only application can never be sure of completely avoiding surrounding non-lesional skin and thus the potential for contamination of the lesional sample with non-lesional skin cannot be completely eliminated. While this size limitation could be partially overcome by supplying tape in a variety of sizes and shapes, this solution would lead to highly inefficient use of tape.